Ling Zhi-8 Research Articles

Polysaccharides PS-G and Protein LZ-8 from Reishi (Ganoderma lucidum) Exhibit Diverse Functions in Regulating Murine Macrophages and T Lymphocytes

Bioactive components in Ganoderma lucidum mainly include polysaccharides (PS-G) and immunomodulatory protein Ling Zhi-8 (LZ-8). These components may have diverse regulatory functions in the immune system. However, the PS-G preparations from different procedures still contained partial LZ-8 residue, indicating that the specific target and regulating function of PS-G and LZ-8 were not fully understood. In the present study, PS-G was subjected to 15% TCA for removing proteins and the LZ-8 detection using anti-LZ-8 monoclonal antibodies showed a remarkable 89.7% protein reduction of the deproteinized PS-G (dpPS-G). The Saccharomyces cerevisiae which expressed recombinant LZ-8 protein (rLZ-8) without glycosylation was generated and then compared with dpPS-G in the induction toward murine primary macrophage and T lymphocytic cells. The peritoneal macrophages from TLR4-deficient and wild type mice revealed that TLR4 was a putative receptor of dpPS-G, mediating the TNF-alpha, IL-1beta and IL-12p70 cytokine production and CD86, MHC II expression on macrophages, while rLZ-8 enhanced the production of IL-1beta, IL-12p70, CD86, and MHC II expression by another obscure route. rLZ-8-treated macrophages enhanced the release of IFN-gamma and IL-2 by murine CD4(+) and CD8(+) T cells, whereas dpPS-G treatment did not enhance the release of IFN-gamma and IL-2. Furthermore, although the direct rLZ-8-treatment conduced dramatic CD154, CD44 expression on CD3(+) T cells and increased IL-2, IFN-gamma secretion on CD4(+) and CD8(+) T cells, the dpPS-G was incapable of priming CD3(+), CD4(+) or CD8(+) T cells unitarily. Taken together, these results demonstrated that LZ-8 could activate murine macrophages and T lymphocytes but PS-G was merely the activator for macrophages, suggesting their diverse roles in activating the innate and adaptive immunity.

An immunomodulatory protein, Ling Zhi-8, induced activation and maturation of human monocyte-derived dendritic cells by the NF-κB and MAPK pathways

Ganoderma lucidum, an oriental medicinal mushroom, has been widely used in Asia to promote health and longevity. LZ-8 is a protein derived from the fungus G. lucidum and has immunomodulatory capacities. In this study, we investigated the immune modulatory effects of rLZ-8 on human monocyte-derived DCs. Treatment of DC with rLZ-8 resulted in the enhanced cell-surface expression of CD80, CD86, CD83, and HLA-DR, as well as the enhanced production of IL-12 p40, IL-10, and IL-23, and the capacity for endocytosis was suppressed in DCs. In addition, treatment of DCs with rLZ-8 resulted in an enhanced, naïve T cell-stimulatory capacity and increased, naïve T cell secretion of IFN-gamma and IL-10. Neutralization with antibodies against TLR4 inhibited the rLZ-8-induced production of IL-12 p40 and IL-10 in DCs. rLZ-8 can stimulate TLR4 or TLR4/MD2-transfected HEK293 cells to produce IL-8. These results suggested an important role for TLR4 in signaling DCs upon incubation with rLZ-8. Further study showed that rLZ-8 was able to augment IKK, NF-kappaB activity, and also IkappaBalpha and MAPK phosphorylation. Further, inhibition of NF-kappaB by helenalin prevented the effects of rLZ-8 in the expression of CD80, CD86, CD83, and HLA-DR and production of IL-12 p40 and IL-10 in various degrees. To confirm the in vitro data, we investigated the effect of rLZ-8 further on antigen-specific antibody and cytokine production in BALB/c mice. Immunization with OVA/rLZ-8 showed that the anti-OVA IgG2a, IFN-gamma, and IL-2 were increased significantly compared with OVA alone in BALB/c mice. In conclusion, our experiments demonstrated that rLZ-8 can effectively promote the activation and maturation of immature DCs, preferring a Th1 response, suggesting that rLZ-8 may possess a potential effect in regulating immune responses.

Reishi immuno‐modulation protein induces interleukin‐2 expression via protein kinase‐dependent signaling pathways within human T cells

Ganoderma lucidum, a medicinal fungus is thought to possess and enhance a variety of human immune functions. An immuno-modulatory protein, Ling Zhi-8 (LZ-8) isolated from G. lucidum exhibited potent mitogenic effects upon human peripheral blood lymphocytes (PBL). However, LZ-8-mediated signal transduction in the regulation of interleukin-2 (IL-2) gene expression within human T cells is largely unknown. Here we cloned the LZ-8 gene of G. lucidum, and expressed the recombinant LZ-8 protein (rLZ-8) by means of a yeast Pichia pastoris protein expression system. We found that rLZ-8 induces IL-2 gene expression via the Src-family protein tyrosine kinase (PTK), via reactive oxygen species (ROS), and differential protein kinase-dependent pathways within human primary T cells and cultured Jurkat T cells. In essence, we have established the nature of the rLZ-8-mediated signal-transduction pathways, such as PTK/protein kinase C (PKC)/ROS, PTK/PLC/PKCalpha/ERK1/2, and PTK/PLC/PKCalpha/p38 pathways in the regulation of IL-2 gene expression within human T cells. Our current results of analyzing rLZ-8-mediated signal transduction in T cells might provide a potential application for rLZ-8 as a pharmacological immune-modulating agent.

Immunomodulating Activities of Ganoderma, a Mushroom with Medicinal Properties

This review highlights the recent research progress in the study of immunomodulating effects of Ganoderma, which belongs to the basidiomycotina class of fungi. The commonly used Ganoderma species include G. lucidum, G. tsugae, G. capense, and G. applanatum. Several major substances with potent immunomodulating action have been isolated from Ganoderma. These include polysaccharides (in particular β-d-glucan), proteins (e.g., Ling Zhi-8) and triterpenoids. The major immunomodulating effects of these active substances derived from Ganoderma include mitogenicity and activation of immune effector cells, such as lymphocytes, macrophages, and natural killer cells, resulting in the production of cytokines, including interleukins (ILs), tumor necrosis factor (TNF)-α, and interferons. There is evidence indicating that β-d-glucans from medicinal mushrooms induce biological response by binding to membrane complement receptor type three (CR3, αMβ2 integrin, or CD11b/CD18) on immune effector cells. The ligand–receptor complex is then internalized, intriguing a series of molecular events such as the activation of the nuclear factor NF-κB. Immunosuppressive effects by Ganoderma components have also been observed. The therapeutic effects of Ganoderma, such as anticancer and anti-inflammatory effects, have been associated with its immunomodulating effects. However, further studies are needed to determine the molecular mechanisms of the immunomodulating effects of Ganoderma mushrooms.

LING ZHI-8

Ling Zhi-8 (LZ-8) is a protein derived from the fungus Ganoderma lucidum and has immunomodulatory capacities. It was shown to be mitogenic toward mouse splenocytes in vitro and immunosuppressive in vivo by reducing antigen-induced antibody formation and by preventing completely the incidence of autoimmune diabetes in nonobese diabetic mice. In this study, the mitogenic effects of LZ-8 on human mononuclear cells are reported. In accordance to its mitogenic effect on mouse splenocytes, LZ-8 proved to be mitogenic for human PBMC. This mitogenic effect of LZ-8 apparently required the presence of monocytes. We also demonstrated it to be immunosuppressive in vitro in a human MLC performed in the absence of monocytes, using purified T cells and EBV-transformed allogeneic B cells. Furthermore, we tested LZ-8 for its possible suppressive effects in 2 different models of allogeneic tissue transplantation. LZ-8 proved to have a significant effect on cellular immunity, since its administration in an allografted mouse skin model resulted in an increased survival time. In a model of transplanted allogeneic pancreatic rat islets, LZ-8 was effective in delaying the rejection process of allografted islets. More frequent or continuous administration resulted in a further prolongation of survival time. No serious side effects of LZ-8 could be discerned in these experiments.

Expression of adhesion molecules in endothelial cells-implication of diabetic vascular dysfunction

Ling Zhi-8 (LZ-8), a novel immunomodulatory protein, markedly enhanced the expression of CDllb, but not CDlla, CD13, CD14, CD18, CD33 or HLA-DR, on the U937 cell line in a dose-dependent fashion. It also induced ICAM-1 expression on vascular endothelial cells and significantly augmented γ -interferon-induced cellular binding between vascular endothelial cells and U937. Furthermore, LZ-8 increased the expression of CD2, but not VLA4, VLA5 or LFA3, on MOLT4 and enhanced rosette formation between human T cells and sheep red blood cells. These data suggest that LZ-8 exerts its pharmacological effect by modulating adhesion molecules on immunocompetent cells.

Ling Zhi-8: A Novel T Cell Mitogen Induces Cytokine Production and Upregulation of ICAM-1 Expression

Ling Zhi-8 (LZ-8) is a protein purified from Ganoderma lucidium, a Chinese medicinal fungus thought to possess potent effects on the immune system. When examined for its effects on lymphocytes, LZ-8 exhibited potent mitogenic effects on human peripheral blood lymphocytes (PBL), inducing a bell-shaped dose-response curve similar to that caused by PHA and other lectin mitogens. Fractionation experiments indicated that the proliferative response in the PBL cultures was primarily due to T cells, but was monocyte dependent. Stimulation of PBL with LZ-8 resulted in the production of IL-2 and a corresponding up regulation of IL-2 receptor expression. In addition to T cell proliferation, microscopic examination of LZ-8-stimulated PBL revealed that LZ-8 induced cellular aggregate formation. The aggregate formation correlated with a dramatic rise in ICAM-1 expression and an increased production of IFN-γ TNFα, and IL-β, molecules associated with regulation of ICAM-1 expression. Both the aggregate formation and the proliferative effects of LZ-8 were blocked by addition of monoclonal antibody to either CD18 or CD11a, the counterreceptor complex components for ICAM-1. Furthermore, addition of neutralizing antibodies to both IL-2 receptor and TNFα blocked aggregate formation, cellular proliferation. and ICAM-1 expression. These findings demonstrate that LZ-8 is a potent T cell activator, mediating its effects via cytokine regulation of integrin expression.

An immunomodulatory protein, ling ZHI-8, facilitates cellular interaction through modulation of adhesion molecules

Ling Zhi-8 (LZ-8), a novel immunomodulatory protein, markedly enhanced the expression of CD11b, but not CD11a, CD13, CD14, CD18, CD33 or HLA-DR, on the U937 cell line in a dose-dependent fashion. It also induced ICAM-1 expression on vascular endothelial cells and significantly augmented gamma - interferon-induced cellular binding between vascular endothelial cells and U937. Furthermore, LZ-8 increased the expression of CD2, but not VLA4, VLA5 or LFA3, on MOLT4 and enhanced rosette formation between human T cells and sheep red blood cells. These data suggest that LZ-8 exerts its pharmacological effect by modulating adhesion molecules on immunocompetent cells.

Molecular cloning of a cDNA and a gene encoding an immunomodulatory protein, Ling Zhi-8, from a fungus, Ganoderma lucidum

A large amount of the novel immunomodulatory protein Ling Zhi-8 (LZ-8) is synthesized in the mycelia of Ganoderma lucidum (Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., and Tsunoo, H. (1989) J. Biol. Chem. 264, 472-478). A cDNA and a gene for LZ-8 were isolated and characterized. The mixed oligonucleotide probes for LZ-8 cDNA were designed from the results of protein sequencing (Tanaka, S., Ko, K., Kino, K., Tsuchiya, K., Yamashita, A., Murasugi, A., Sakuma, S., and Tsunoo, H. (1989) J. Biol. Chem. 264, 16372-16377) and were used for screening the mycelial cDNA library. The nucleotide sequence of the cloned cDNA confirms the amino acid sequence of LZ-8 that was previously determined by protein sequencing. The clones containing the LZ-8 gene (lz-8) were obtained from the mycelial genomic DNA library using the cDNA probe. Two CCAAT-like sequences and one TATA box were found at the upstream region of the postulated transcription initiation site of lz-8. A small intron (61 nucleotides long) divided lz-8 into two exons at the 5'-untranslated region. The other characteristic sequences were also found around the postulated transcription initiation site and around the poly(A) additional site.

An immunomodulating protein, Ling Zhi-8 (LZ-8) prevents insulitis in non-obese diabetic mice

Ling Zhi-8 (LZ-8), a novel and recently discovered immunomodulatory protein having in vivo immuno-suppressive activity, was tested for in vivo effect against Type 1 (insulin-dependent) diabetes mellitus in the nonobese diabetic mouse, the disease having immunologically mediated aetiology in this animal. LZ-8 had mitogenic activity in vitro towards spleen cells of the non-obese diabetic mice as previously shown towards those of DBA/2 mice. Intraperitoneal administration of LZ-8 twice weekly into the mice (10.3-12.6 mg/kg body weight) from 4 weeks of age prevented insulitis and an almost normal number of insulin producing cells were observed. Extreme insulitis and reduction of the number of insulin producing cells were observed in the pancreata of the untreated non-obese diabetic mouse. No cumulative incidence of diabetes mellitus was observed in the LZ-8 treated group, while cumulative incidences of 70% and 60% were observed in an untreated group followed up to 42 weeks of age when the incidence of diabetes was defined as a plasma glucose level of greater than 11 mmol/l and as a urine glucose level of greater than 2+, respectively. T cell subset population analysis was performed to further investigate the action of LZ-8 on the non-obese diabetic mouse which revealed that LZ-8 treatment increased in L3T4'/Lyt-2+ ratio.

Complete amino acid sequence of an immunomodulatory protein, ling zhi-8 (LZ-8): An immunomodulator from a fungus, Ganoderma lucidium, having similarity to immunoglobulin variable regions

The complete amino acid sequence of a novel immunomodulatory protein, ling zhi-8 (LZ-8), isolated from a fungus, Ganoderma lucidium (Kino, K., Yamashita, A., Yamaoka, K., Watanabe, J., Tanaka, S., Ko, K., Shimizu, K., and Tsunoo, H. (1989) J. Biol. Chem. 264, 472-478), was determined by protein sequencing. The polypeptide consists of 110 amino acid residues with an acetylated amino end and has a molecular mass of 12,420 Da including an amino-end blocking group. There is no attachment site for an Asn-linked oligosaccharide chain, consistent with the very low carbohydrate content of LZ-8. These results indicate that the native form of LZ-8 with a molecular mass of 24 kDa is a homodimer of the LZ-8 polypeptide whose sequence is described here. Furthermore, the LZ-8 chain shows considerable similarity to the variable region of immunoglobulin heavy chain both in its sequence and in its predicted secondary structure. The interesting possibility that LZ-8 is related to an ancestral protein of the immunoglobulin superfamily is also discussed.

Isolation and Characterization of a New Immunomodulatory Protein, Ling Zhi-8 (LZ-8), from Ganoderma lucidium

A novel protein with mitogenic activity in vitro and immunomodulating activity in vivo has been isolated from the mycelial extract of an Oriental medicinal fungus, ling zhi (Ganoderma lucidium). This protein was named ling zhi-8 (LZ-8) and its biochemical and immunological properties are described. LZ-8 was purified by two chromatographic systems, gel filtration and followed by ion-exchange, using an in vitro bioassay measuring blast-formation stimulatory activity toward mouse spleen lymphocytes to monitor purification. Analysis by several types of electrophoresis revealed a single band, with the molecular weight differing slightly depending on the system employed. Under reduced conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the method of Laemmli, U.K. ((1970) Nature 227, 680-685) indicated an apparent Mr = 17,100, while under nonreduced conditions an apparent Mr = 17,500 was found; and, using Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a value of apparent Mr = 13,100 was obtained. LZ-8 has an isoelectric point of 4.4, and sugar analysis indicated a low carbohydrate content (1.3%). Half-cysteine, histidine, and methionine were not detected from the analysis of amino acid composition after further purification of LZ-8 by reversed-phase high performance liquid chromatography. LZ-8 was capable of hemagglutinating sheep red blood cells, but no such activity was observed toward human red blood cells (A, B, AB, and O types). In vivo, LZ-8 prevents the production of systemic anaphylaxis reaction in mice if it has been administered repeatedly, and reduction of antibody production is the suggested mechanism. The mechanisms of hemagglutination of sheep red blood cells and of blast-formation stimulation of mouse spleen cells are also discussed.